RESUMEN
The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Anti-VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/química , VIH-1/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
VRC01-class antibodies neutralize diverse HIV-1 strains by targeting the conserved CD4-binding site. Despite extensive investigations, crucial events in the early stage of VRC01 development remain elusive. We demonstrated how VRC01-class antibodies emerged in a Chinese donor by antigen-specific single B cell sorting, structural and functional studies, and longitudinal antibody and virus repertoire analyses. A monoclonal antibody DRVIA7 with modest neutralizing breadth was isolated that displayed a subset of VRC01 signatures. X-ray and EM structures revealed a VRC01-like angle of approach, but less favorable interactions between the DRVIA7 light-chain CDR1 and the N terminus with N276 and V5 glycans of gp120. Although the DRVIA7 lineage was unable to acquire broad neutralization, longitudinal analysis revealed a repertoire-encoded VRC01 light-chain CDR3 signature and VRC01-like neutralizing heavy-chain precursors that rapidly matured within 2 years. Thus, light chain accommodation of the glycan shield should be taken into account in vaccine design targeting this conserved site of vulnerability.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Sitios de Unión de Anticuerpos/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Anticuerpos ampliamente neutralizantes , Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Datos de Secuencia MolecularRESUMEN
Many neutralizing antibodies (nAbs) elicited to ancestral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through natural infection and vaccination have reduced effectiveness to SARS-CoV-2 variants. Here, we show that therapeutic antibody ADG20 is able to neutralize SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize a broad range of VOCs, albeit with reduced potency against Omicron. Thus, this conserved and vulnerable site can be exploited for the design of universal vaccines and therapeutic antibodies.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , COVID-19/virología , Epítopos/inmunología , Humanos , Pruebas de Neutralización , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
The high-mannose patch on the HIV-1 envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet some variation evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provides insights to advance design of HIV vaccine candidates.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos/genética , Antígenos Virales/química , Antígenos Virales/inmunología , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , Epítopos/química , Células HEK293 , Anticuerpos Anti-VIH/química , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica de Transmisión , Mutagénesis Sitio-Dirigida , Polisacáridos/inmunología , Hipermutación Somática de Inmunoglobulina , Proteínas del Envoltorio Viral/inmunología , Difracción de Rayos X , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.
Asunto(s)
Proteínas de la Cápside/química , VIH-1/química , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , VIH-1/metabolismo , Modelos Moleculares , Polímeros/metabolismo , Estructura Terciaria de ProteínaRESUMEN
A key challenge in the quest toward an HIV-1 vaccine is design of immunogens that can generate a broadly neutralizing antibody (bnAb) response against the enormous sequence diversity of the HIV-1 envelope glycoprotein (Env). We previously demonstrated that a recombinant, soluble, fully cleaved SOSIP.664 trimer based on the clade A BG505 sequence is a faithful antigenic and structural mimic of the native trimer in its prefusion conformation. Here, we sought clade C native-like trimers with comparable properties. We identified DU422 and ZM197M SOSIP.664 trimers as being appropriately thermostable (Tm of 63.4 °C and 62.7 °C, respectively) and predominantly native-like, as determined by negative-stain electron microscopy (EM). Size exclusion chromatography, ELISA, and surface plasmon resonance further showed that these trimers properly display epitopes for all of the major bnAb classes, including quaternary-dependent, trimer-apex (e.g., PGT145) and gp120/gp41 interface (e.g., PGT151) epitopes. A cryo-EM reconstruction of the ZM197M SOSIP.664 trimer complexed with VRC01 Fab against the CD4 binding site at subnanometer resolution revealed a striking overall similarity to its BG505 counterpart with expected local conformational differences in the gp120 V1, V2, and V4 loops. These stable clade C trimers contribute additional diversity to the pool of native-like Env immunogens as key components of strategies to induce bnAbs to HIV-1.
Asunto(s)
Diseño de Fármacos , VIH-1/inmunología , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Microscopía por Crioelectrón , Antígenos VIH/química , Antígenos VIH/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Moleculares , Coloración Negativa , Estabilidad Proteica , Solubilidad , TemperaturaRESUMEN
Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named "PGDM1400-1412," show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC50 of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Secuencia de Aminoácidos , Linfocitos B/inmunología , Secuencia de Bases , Reacciones Cruzadas , Epítopos/química , Variación Genética , Infecciones por VIH/inmunología , Humanos , Memoria Inmunológica , Concentración 50 Inhibidora , Leucocitos Mononucleares/citología , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Pruebas de Neutralización , Biblioteca de Péptidos , Filogenia , Pliegue de Proteína , Proteínas Recombinantes/inmunología , Homología de Secuencia de AminoácidoRESUMEN
The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6â Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.
Asunto(s)
Anticuerpos Neutralizantes/química , Epítopos/química , VIH-1/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Anticuerpos Neutralizantes/inmunología , Cristalografía por Rayos X , Epítopos/inmunología , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Modelos Moleculares , Polisacáridos/química , Polisacáridos/inmunología , Conformación Proteica , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
Many neutralizing antibodies (nAbs) elicited to ancestral SARS-CoV-2 through natural infection and vaccination generally have reduced effectiveness to SARS-CoV-2 variants. Here we show therapeutic antibody ADG20 is able to neutralize all SARS-CoV-2 variants of concern (VOCs) including Omicron (B.1.1.529) as well as other SARS-related coronaviruses. We delineate the structural basis of this relatively escape-resistant epitope that extends from one end of the receptor binding site (RBS) into the highly conserved CR3022 site. ADG20 can then benefit from high potency through direct competition with ACE2 in the more variable RBS and interaction with the more highly conserved CR3022 site. Importantly, antibodies that are able to target this site generally neutralize all VOCs, albeit with reduced potency against Omicron. Thus, this highly conserved and vulnerable site can be exploited for design of universal vaccines and therapeutic antibodies.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Beta variant of concern (VOC) resists neutralization by major classes of antibodies from COVID-19 patients and vaccinated individuals. In this study, serum of Beta-infected patients revealed reduced cross-neutralization of wild-type virus. From these patients, we isolated Beta-specific and cross-reactive receptor-binding domain (RBD) antibodies. The Beta-specificity results from recruitment of VOC-specific clonotypes and accommodation of mutations present in Beta and Omicron into a major antibody class that is normally sensitive to these mutations. The Beta-elicited cross-reactive antibodies share genetic and structural features with wild type-elicited antibodies, including a public VH1-58 clonotype that targets the RBD ridge. These findings advance our understanding of the antibody response to SARS-CoV-2 shaped by antigenic drift, with implications for design of next-generation vaccines and therapeutics.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/inmunología , Reacciones Cruzadas , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/metabolismo , Deriva y Cambio Antigénico , COVID-19/virología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3-53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3-53 neutralizing antibodies +/- Fab CR3022 ranging from 2.33 to 3.11 Å resolution. The germline-encoded residues of IGHV3-53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3-53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3-53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.
RESUMEN
To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the "neutralizing face" on E2, including several broadly nAbs encoded by the VH1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2-VH1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.
Asunto(s)
Hepatitis C , Vacunas contra Hepatitis Viral , Anticuerpos Neutralizantes , Epítopos , Hepacivirus , Anticuerpos contra la Hepatitis C , HumanosRESUMEN
Molecular understanding of neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could accelerate vaccine design and drug discovery. We analyzed 294 anti-SARS-CoV-2 antibodies and found that immunoglobulin G heavy-chain variable region 3-53 (IGHV3-53) is the most frequently used IGHV gene for targeting the receptor-binding domain (RBD) of the spike protein. Co-crystal structures of two IGHV3-53-neutralizing antibodies with RBD, with or without Fab CR3022, at 2.33- to 3.20-angstrom resolution revealed that the germline-encoded residues dominate recognition of the angiotensin I converting enzyme 2 (ACE2)-binding site. This binding mode limits the IGHV3-53 antibodies to short complementarity-determining region H3 loops but accommodates light-chain diversity. These IGHV3-53 antibodies show minimal affinity maturation and high potency, which is promising for vaccine design. Knowledge of these structural motifs and binding mode should facilitate the design of antigens that elicit this type of neutralizing response.
Asunto(s)
Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Formación de Anticuerpos , Betacoronavirus/inmunología , Regiones Determinantes de Complementariedad/química , Infecciones por Coronavirus/prevención & control , Cadenas Pesadas de Inmunoglobulina/química , Pandemias/prevención & control , Neumonía Viral/prevención & control , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Sitios de Unión , COVID-19 , Vacunas contra la COVID-19 , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Cristalografía por Rayos X , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Neumonía Viral/inmunología , Dominios Proteicos , SARS-CoV-2 , Vacunas Virales/química , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
An effective vaccine to the antigenically diverse hepatitis C virus (HCV) must target conserved immune epitopes. Here, we investigate cross-neutralization of HCV genotypes by broadly neutralizing antibodies (bNAbs) encoded by the relatively abundant human gene family V H 1-69. We have deciphered the molecular requirements for cross-neutralization by this unique class of human antibodies from crystal structures of HCV E2 in complex with bNAbs. An unusually high binding affinity is found for germ line-reverted versions of VH1-69 precursor antibodies, and neutralization breadth is acquired during affinity maturation. Deep sequencing analysis of an HCV-immune B cell repertoire further demonstrates the importance of the V H 1-69 gene family in the generation of HCV bNAbs. This study therefore provides critical insights into immune recognition of HCV with important implications for rational vaccine design.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/inmunología , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Hepatitis C/inmunología , Afinidad de Anticuerpos/inmunología , Sitios de Unión , Donantes de Sangre , Línea Celular Tumoral , Reacciones Cruzadas/inmunología , Epítopos/química , Hepatitis C/virología , Humanos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Vacunas contra Hepatitis Viral/genética , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
Stabilized HIV-1 envelope glycoproteins (Env) that resemble the native Env are utilized in vaccination strategies aimed at inducing broadly neutralizing antibodies (bNAbs). To limit the exposure of rare isolate-specific antigenic residues/determinants we generated a SOSIP trimer based on a consensus sequence of all HIV-1 group M isolates (ConM). The ConM trimer displays the epitopes of most known bNAbs and several germline bNAb precursors. The crystal structure of the ConM trimer at 3.9 Å resolution resembles that of the native Env trimer and its antigenic surface displays few rare residues. The ConM trimer elicits strong NAb responses against the autologous virus in rabbits and macaques that are significantly enhanced when it is presented on ferritin nanoparticles. The dominant NAb specificity is directed against an epitope at or close to the trimer apex. Immunogens based on consensus sequences might have utility in engineering vaccines against HIV-1 and other viruses.
Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Epítopos/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Secuencia de Consenso , Macaca , Multimerización de Proteína , ConejosRESUMEN
Induction of broadly neutralizing antibodies (bNAbs) by HIV-1 envelope glycoprotein immunogens would be a major advance toward an effective vaccine. A critical step in this process is the activation of naive B cells expressing germline (gl) antibody precursors that have the potential to evolve into bNAbs. Here, we reengineered the BG505 SOSIP.664 glycoprotein to engage gl precursors of bNAbs that target either the trimer apex or the CD4-binding site. The resulting BG505 SOSIP.v4.1-GT1 trimer binds multiple bNAb gl precursors in vitro. Immunization experiments in knock-in mice expressing gl-VRC01 or gl-PGT121 show that this trimer activates B cells in vivo, resulting in the secretion of specific antibodies into the sera. A crystal structure of the gl-targeting trimer at 3.2-Å resolution in complex with neutralizing antibodies 35O22 and 9H+109L reveals a native-like conformation and the successful incorporation of design features associated with binding of multiple gl-bNAb precursors.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Proteínas gp160 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Animales , Cristalografía por Rayos X , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Ratones , Multimerización de Proteína/inmunología , Estructura Terciaria de ProteínaRESUMEN
Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli. A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E. coli strains with additional tRNA genes to supplement sparsely used E. coli codons have been most successful. The expressed proteins have a wide range of molecular weights and number of transmembrane helices. Expression of these proteins has been observed in the membrane and insoluble fraction of E. coli cell lysates and, in some cases, in the soluble fraction. The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices. In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.
Asunto(s)
Escherichia coli/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Mycobacterium tuberculosis/genética , Clonación Molecular , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Membranas Intracelulares/metabolismoRESUMEN
Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold ß sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.
Asunto(s)
Proteínas del Envoltorio Viral/química , Anticuerpos Neutralizantes/química , Antivirales/química , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Epítopos/química , Epítopos/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Estructura Terciaria de Proteína , Tetraspanina 28/química , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/química , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan-dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield and access the gp120 protein surface. EM reveals that PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. Combined structural studies of PGT 135, PGT 128 and 2G12 show that this Asn332-dependent antigenic region is highly accessible and much more extensive than initially appreciated, which allows for multiple binding modes and varied angles of approach; thereby it represents a supersite of vulnerability for antibody neutralization.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Alcaloides/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/metabolismo , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos , Biopolímeros , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Secuencia de Carbohidratos , Cristalografía por Rayos X , Epítopos/química , Epítopos/inmunología , Glicosilación/efectos de los fármacos , Células HEK293 , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/ultraestructura , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Microscopía Electrónica , Modelos Moleculares , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Polisacáridos/fisiología , Conformación Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Relación Estructura-Actividad , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
The human immunodeficiency virus type 1 capsid is modeled as a fullerene cone that is composed of approximately 250 hexamers and 12 pentamers of the viral CA protein. Structures of CA hexamers have been difficult to obtain because the hexamer-stabilizing interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Here, we describe a two-step biochemical strategy to obtain soluble CA hexamers for crystallization. First, the hexamer was stabilized by engineering disulfide cross-links (either A14C/E45C or A42C/T54C) between the N-terminal domains of adjacent subunits. Second, the cross-linked hexamers were prevented from polymerizing further into hyperstable capsid-like structures by mutations (W184A and M185A) that interfered with dimeric association between the C-terminal domains that link adjacent hexamers. The structures of two different cross-linked CA hexamers were nearly identical, and we combined the non-mutated portions of the structures to generate an atomic resolution model for the native hexamer. This hybrid approach for structure determination should be applicable to other viral capsomers and protein-protein complexes in general.